Processing of the phalloidin proprotein by prolyl oligopeptidase from the mushroom Conocybe albipes

Hong Luo, Heather E Hallen-Adams, Jonathan D. Walton

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (phallotoxins). In Amanita bisporigera, α-amanitin and phallacidin are synthesized as 35- and 34-amino acid proproteins, respectively, in which the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences. The presence of invariant Pro residues immediately upstreamm of the toxin regions and as the last predicted amino acid in-the toxin regions themselves suggests that a Pro-specific peptidase is responsible for the initial post-translational processing of the Amanita toxin proproteins. We purified an enzyme from the phalloidin-producing mushroom Conocybe albipes that cleaves a synthetic 22-mer phalloidin peptide to release the mature toxin peptide (AWLATCP). Mass spectrometric analysis of the purified protein combined with isolation and sequencing of the encoding gene indicates that the responsible processing enzyme is a member of the prolyl oligopeptidase (POP) subfamily of proteases (EC 3.4.21.26). The processing enzyme was able to use the chromogenic POP substrate benzyloxycarbonyl-Gly-Pro-p-nitro-anilide and was inhibited by the specific POP inhibitor benzyl-oxycarbonyl-Pro-prolinal. Both Pro bonds in the proprotein are cleaved by the same enzyme, with the C-terminal Pro bond cleaved first or much faster than the N-terminal Pro bond. Transient accumulation of the N-terminal intermediate indicates that cleavage is not strongly processive. A synthetic peptide representing the phallacidin proprotein was also cleaved by the POP of C. albipes, but a precursor of amanitin (which is not made by C. albipes) was cleaved inefficiently.

Original languageEnglish (US)
Pages (from-to)18070-18077
Number of pages8
JournalJournal of Biological Chemistry
Volume284
Issue number27
DOIs
StatePublished - Jul 3 2009

Fingerprint

prolyl oligopeptidase
Phalloidine
Agaricales
Amanita
Amanitins
Amino Acids
Peptides
Enzymes
Processing
Peptide Hydrolases
Anilides
Chromogenics
Amino Acid Sequence
Gene encoding
Substrates
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Processing of the phalloidin proprotein by prolyl oligopeptidase from the mushroom Conocybe albipes. / Luo, Hong; Hallen-Adams, Heather E; Walton, Jonathan D.

In: Journal of Biological Chemistry, Vol. 284, No. 27, 03.07.2009, p. 18070-18077.

Research output: Contribution to journalArticle

@article{28547ee0bd7d416b9b6f89b229a942ea,
title = "Processing of the phalloidin proprotein by prolyl oligopeptidase from the mushroom Conocybe albipes",
abstract = "The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (phallotoxins). In Amanita bisporigera, α-amanitin and phallacidin are synthesized as 35- and 34-amino acid proproteins, respectively, in which the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences. The presence of invariant Pro residues immediately upstreamm of the toxin regions and as the last predicted amino acid in-the toxin regions themselves suggests that a Pro-specific peptidase is responsible for the initial post-translational processing of the Amanita toxin proproteins. We purified an enzyme from the phalloidin-producing mushroom Conocybe albipes that cleaves a synthetic 22-mer phalloidin peptide to release the mature toxin peptide (AWLATCP). Mass spectrometric analysis of the purified protein combined with isolation and sequencing of the encoding gene indicates that the responsible processing enzyme is a member of the prolyl oligopeptidase (POP) subfamily of proteases (EC 3.4.21.26). The processing enzyme was able to use the chromogenic POP substrate benzyloxycarbonyl-Gly-Pro-p-nitro-anilide and was inhibited by the specific POP inhibitor benzyl-oxycarbonyl-Pro-prolinal. Both Pro bonds in the proprotein are cleaved by the same enzyme, with the C-terminal Pro bond cleaved first or much faster than the N-terminal Pro bond. Transient accumulation of the N-terminal intermediate indicates that cleavage is not strongly processive. A synthetic peptide representing the phallacidin proprotein was also cleaved by the POP of C. albipes, but a precursor of amanitin (which is not made by C. albipes) was cleaved inefficiently.",
author = "Hong Luo and Hallen-Adams, {Heather E} and Walton, {Jonathan D.}",
year = "2009",
month = "7",
day = "3",
doi = "10.1074/jbc.M109.006460",
language = "English (US)",
volume = "284",
pages = "18070--18077",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "27",

}

TY - JOUR

T1 - Processing of the phalloidin proprotein by prolyl oligopeptidase from the mushroom Conocybe albipes

AU - Luo, Hong

AU - Hallen-Adams, Heather E

AU - Walton, Jonathan D.

PY - 2009/7/3

Y1 - 2009/7/3

N2 - The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (phallotoxins). In Amanita bisporigera, α-amanitin and phallacidin are synthesized as 35- and 34-amino acid proproteins, respectively, in which the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences. The presence of invariant Pro residues immediately upstreamm of the toxin regions and as the last predicted amino acid in-the toxin regions themselves suggests that a Pro-specific peptidase is responsible for the initial post-translational processing of the Amanita toxin proproteins. We purified an enzyme from the phalloidin-producing mushroom Conocybe albipes that cleaves a synthetic 22-mer phalloidin peptide to release the mature toxin peptide (AWLATCP). Mass spectrometric analysis of the purified protein combined with isolation and sequencing of the encoding gene indicates that the responsible processing enzyme is a member of the prolyl oligopeptidase (POP) subfamily of proteases (EC 3.4.21.26). The processing enzyme was able to use the chromogenic POP substrate benzyloxycarbonyl-Gly-Pro-p-nitro-anilide and was inhibited by the specific POP inhibitor benzyl-oxycarbonyl-Pro-prolinal. Both Pro bonds in the proprotein are cleaved by the same enzyme, with the C-terminal Pro bond cleaved first or much faster than the N-terminal Pro bond. Transient accumulation of the N-terminal intermediate indicates that cleavage is not strongly processive. A synthetic peptide representing the phallacidin proprotein was also cleaved by the POP of C. albipes, but a precursor of amanitin (which is not made by C. albipes) was cleaved inefficiently.

AB - The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (phallotoxins). In Amanita bisporigera, α-amanitin and phallacidin are synthesized as 35- and 34-amino acid proproteins, respectively, in which the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences. The presence of invariant Pro residues immediately upstreamm of the toxin regions and as the last predicted amino acid in-the toxin regions themselves suggests that a Pro-specific peptidase is responsible for the initial post-translational processing of the Amanita toxin proproteins. We purified an enzyme from the phalloidin-producing mushroom Conocybe albipes that cleaves a synthetic 22-mer phalloidin peptide to release the mature toxin peptide (AWLATCP). Mass spectrometric analysis of the purified protein combined with isolation and sequencing of the encoding gene indicates that the responsible processing enzyme is a member of the prolyl oligopeptidase (POP) subfamily of proteases (EC 3.4.21.26). The processing enzyme was able to use the chromogenic POP substrate benzyloxycarbonyl-Gly-Pro-p-nitro-anilide and was inhibited by the specific POP inhibitor benzyl-oxycarbonyl-Pro-prolinal. Both Pro bonds in the proprotein are cleaved by the same enzyme, with the C-terminal Pro bond cleaved first or much faster than the N-terminal Pro bond. Transient accumulation of the N-terminal intermediate indicates that cleavage is not strongly processive. A synthetic peptide representing the phallacidin proprotein was also cleaved by the POP of C. albipes, but a precursor of amanitin (which is not made by C. albipes) was cleaved inefficiently.

UR - http://www.scopus.com/inward/record.url?scp=67650594524&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=67650594524&partnerID=8YFLogxK

U2 - 10.1074/jbc.M109.006460

DO - 10.1074/jbc.M109.006460

M3 - Article

VL - 284

SP - 18070

EP - 18077

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 27

ER -